The immobilization of functional proteins on flat surfaces is of crucial importance for studying their interaction with ligands and examining their structure by means of electron and scanning probe microscopy and other biophysical techniques requiring a solid interface. Targeting proteins at specific sites and anisotropically immobilizing them on a surface while preserving their functionality is a major precondition to facilitate biochemical recognition and interaction, to present selected sites for structural investigation, to induce two-dimensional crystallization, and to develop new biosensors and supramolecular assemblies.
Generally, two methods have been used to attach (immobilize) proteins to a solid surface. In the first method, the peptide is applied to the surface in solution, which is then evaporated off, leaving the peptide dried to the surface. Such non-specific attachment is inefficient for small peptides and applicable only to methods which do not require a large concentration of immobilized peptide, as much will be resolubilized subsequently in the presence of solution. Moreover, because the attachment is non-specific, peptides will be attached in random and variant orientations. Where presentation of a particular active site is critical, such variance can further reduce the specificity of the bound peptide.
In the more common two-step chemical coupling process, the solid surface is first passively coated with a large protein, such as an immunoglobulin or bovine serum albumin. Then, a hetero-bifunctional cross-linking agent, such as SPDP or glutaraldehyde, is attached to the protein and used to capture peptide from solution. Such a method, while time consuming, is currently used, for example, in cell culture procedures which require a high concentration of bound peptide. While this typically results in higher quantities of bound protein, because a protein may contain numerous sites capable of interacting, peptides will be attached in random and variant orientations. Where presentation of a particular active site is critical, such variance can further reduce the specificity of the bound peptide with the cross-linker. In addition it is often difficult or impossible to remove from the underlying surface.
An alternative technique for binding proteins has been used for protein purification. This method, named “Immobilized Metal Affinity Chromatography” (IMAC) resulted from the recognition that certain proteins have an affinity for heavy metal ions, which could be an additional distinguishing feature to use in attempting separation of the proteins. This feature applies especially to proteins containing histidine or cysteine residues, which have been found to complex with chelated zinc or copper ions and become adsorbed on a chelating resin (Porath et al. (1975) Nature, 258: 598-99 (1975). Again, this method suffers the deficiency that it requires a metal cation coordinated to a surface which is difficult to produce in an atomically smooth form. In addition, metals are toxic to a number of biological processes limiting their efficacy in assays requiring biological activity of one or more components. Finally, the orientation of proteins on metal surfaces is difficult to control.
Several efforts have been made to immobilize proteins with controlled orientation either covalently utilizing single reactive thiol groups of cysteine residues (Colliuod et al. (1993) Bioconjugate Chem. 4, 528-536 ), or non-covalently, but specifically via immobilized antibodies (Schuhmann et al. (1991) Adv. Mater. 3: 388-391; Lu et al. (1995) Anal. Chem. 67: 83-87), the biotin/streptavidin system (Iwane et al. (1997) Biophys. Biochem. Res. Comm. 230: 76-80), or metal-chelating Langmuir-Blodgett films (Ng et al. (1995) Langmuir 11: 4048-4055; Schmitt et al. (1996) Angew. Chem. Int. Ed. Engl. 35: 317-320; Frey et al. (1996) Proc. Natl. Acad. Sci. USA 93: 4937-4941; Kubalek et al. (1994) J. Struct. Biol., 113: 117-123) and metal-chelating self-assembled monolayers (Sigal et al. (1996) Analytical Chem., 68: 490-497) for binding of polyhistidine fusion proteins. Mica, with the ideal structure KAl2[AlSi3O10](OH,F)2, refers to a group of layered aluminosilicate minerals whose crystals exhibit a large degree of basal cleavage, allowing them to be split into very thin atomically flat sheets.
Due to its flatness and hydrophilic surface, mica has been established as a standard substrate for electron and scanning probe microscopy applications (see e.g., Zahn et al. (1993) J. Mol. Biol., 229: 579-584; Yang et al. (1994) FEBS Lett. 338: 89-92; Guckenberger et al. (1994) Science 266: 1538-1540; Mueller et al. (1996) Biophys. J. 70: 1796-1802). Therefore, chemical modification of and site-specific immobilization on mica would extend its field of applications towards more sophisticated molecular architectures.
The complex multilayered structure of mica with its surface-exposed negatively charged honeycomb arrangements of Si(Al)O4 tetrahedra has been used as substrate for monolayer formation of amphiphilic organic molecules, such as alkylphosphonic acids [Woodward et al. (1996) J. Am. Chem. Soc. 118: 7861-7862) and organosilanes (Schwartz, et al. (1992) Phys. Rev. Lett. 69: 3354-3357, Okusa et al. (1994) Langmuir 10: 3577-3581; Hu et al. (1996) Langmuir 12: 1697-1700; Xiao et al. (1996) Langmuir 12: 235-237; Britt et al. (1996) J. Colloid Interface Sci. 178: 775-784). Unfortunately, the former are not robust under aqueous conditions and the latter are often isotropically rough with monolayer formation characterized by low reproducibility, especially if terminated with a nucleophilic group in the w-position.
As an alternative to the attachment of a two-dimensional siloxane network onto the mica surface, efforts have been made to alter the surface chemistry by exchanging the surface cations (mostly potassium) at the basal (001) cleavage plane with other inorganic and organic ions (Shelden et al. (1993) J. Colloid Interface Sci., 157: 318-327; Hähner et al. (1996) J. Chem. Phys. 104: 7749-7757). Surfactant adsorption of long-chain alkylammonium salts, such as cetyltrimethylammonium bromide (CTAB) (Sharma et al. (1996) Langmuir 12: 6506-6512; Eriksson et al. (1996) J. Colloid Interface Sci. 181: 476-489) and N-dodecylpyridinium chloride (NDP) (Shelden et al. (1993) J. Colloid Interface Sci., 157: 318-327) are known to hydrophobize negatively charged minerals. Exchange with bivalent cations has been used to mediate binding of DNA for SPM studies (Hansma et al. (1995) Biophys. J. 68: 1672-1677). Similarly, 2,2′-azobisisobutyramidine hydrochloride (AIBA) has been used as an azo initiator for the polymerization of styrene directly on the mica surface (Shelden et al. (1993) J. Colloid Interface Sci., 157: 318-327; Shelden et al. (1994) Polymer, 35: 1571-1575).
There thus exists a need for a rapid and reproducible one step-process for attaching polypeptides, and other moieties such, to a solid surface. Ideally, such a method should be easy to perform and efficient. In addition, it should preferably result in appropriate presentation of critical epitopes, binding sites, and/or active sites. The present invention satisfies these needs and provides related advantages as well.